Bacterial vaginosis (BV) is a common but highly enigmatic condition that is associated with adverse outcomes for women and their neonates. BV is characterized by shifts in the vaginal microbiota from Lactobacillus dominant to a microbiota with diverse anaerobic bacteria. Small molecule metabolites in the vagina influence host physiology, affect microbial community composition, and impact risk of adverse health outcomes.
The composition of small molecule metabolites in the human vagina is largely shaped by bacterial metabolism of human derived nutrients. These bacterial metabolites may impact human cell function, inflammation, and disease susceptibility. However, few studies have comprehensively examined the associations between vaginal bacteria and metabolites, and no study has used quantitative PCR to link concentrations of fastidious vaginal bacteria to metabolite concentrations. Individual metabolites associated with BV status have been identified and shown to be useful markers of BV such as lactate (no BV) and succinate (BV) (1-7). However, there are hundreds of other compounds that have not been identified in vaginal fluid and linked to particular bacterial communities or clinical outcomes.
The composition of the human vaginal microbiota ranges from communities dominated by a limited of Lactobacillus species to complex communities of diverse anaerobes, most evident with the common condition BV (8-11). BV affects up to 29% of women in the United States (12), and has been associated with several adverse reproductive and health outcomes including elevated risks for acquisition of sexually transmitted infections (13-15), transmission of HIV to male sex partners (16), preterm birth (17), pelvic inflammatory disease (18), and cervicitis (19). The microbiota in BV is heterogeneous; different groups of women have vaginal microbiotas dominated with different bacteria such as BV-associated bacterium-1 (BVAB1), Leptotrichia/Sneathia species, Prevotella spp., Gardnerella vaginalis, or Lactobacillus iners, while others are colonized with diverse bacteria and no single dominant bacterium (11, 20). BV is diagnosed in clinical settings using Amsel criteria in which a set of four signs or symptoms are evaluated, with at least three required for a diagnosis of BV (21). Different bacteria are correlated with each of these four clinical criteria (11). Small molecule metabolites can affect at least three of these clinical criteria including vaginal discharge, pH, and amine odor. The diagnosis of BV can also be made using Gram stain interpretation of vaginal fluid smears with enumeration of bacterial morphotypes (22).
Polyamines such as putrescine and cadaverine are found in women with BV and contribute to the fishy amine odor in this condition (1, 23-25). These polyamines are likely produced from decarboxylation of amino acids mediated by bacteria (23). Chen et al. demonstrated that in vitro production of polyamines by mixed anaerobic vaginal bacteria was inhibited in the presence of metronidazole, suggesting a role for bacterial production of polyamines (26). Likewise, trimethylamine has a fishy odor, is found in women with BV, and is also thought to be a product of bacterial metabolism (27). These amines become volatile when pH is elevated, a property utilized in the clinical diagnosis of BV when employing the “whiff test” (4, 28, 29). The amine odor can also be noted in vivo with elevated pH during menses and after vaginal intercourse with men, as the pH of blood and semen are close to 7 leading to enhanced volatilization. It has been suggested that the amines are associated with increased vaginal transudation of fluid and squamous cell exfoliation, resulting in the thin homogeneous grayish-white discharge that is typically associated with BV (4).
Mass spectrometry (when coupled with chromatography) offers the opportunity to measure small molecule metabolites in the vagina, and assign identity to hundreds of compounds simultaneously. One recent study described the metabolite profiles in eight women with BV using this approach, and classified these women in to two groups based on their vaginal metabolite compositions (30). Additional insights into the functions of these bacterial consortia can be gained by linking bacterial community composition and concentrations of bacteria to metabolic signatures.
Current methods of diagnosing BV have numerous disadvantages. Most common methods of diagnosing BV and their disadvantages currently include: (1) Clinical settings: Amsel clinical criteria which include a set of four signs or symptoms of which at least three are required for a positive diagnosis. These include pH, a positive “whiff test,” greater than 20% clue cells (vaginal epithelial cells coated with bacteria) and a thin homogeneous discharge. This diagnostic procedure requires a trained clinician and a microscope is needed to evaluate clue cells; (2) Research settings: Vaginal fluid Gram stains and evaluation of the morphotypes based on the Nugent method or variations of the Ison & Hay method. The Nugent method is the gold standard in research settings, however, a highly trained microscopist is needed for evaluating the slides; (3) Detection of bacterial nucleic acids: Gen-Probe has developed a test targeting bacterial nucleic acids from bacteria that cause BV. Testing nucleic acids by PCR is typically expensive. Becton Dickinson has a BD Affirm VPIII system that can detect high levels of Gardnerella vaginalis DNA. However, G. vaginalis is not specific for the diagnosis of BV, as it is present in 70% of women without BV; (4) Detection of bacterial sialidase: OSOM BVBlue test that measures bacterial sialidases that are elevated in BV. While one study showed that the test was sensitive and specific (Bradshaw et al., 2005 JCM 43:1304-1308), other studies have not been able to demonstrate high sensitivity (Madhivanan et al., 2014 Infect Dis Obstet Gynecol Article ID 908313; Hillier https://clinicaltrials.gov/ct2/show/results/NCT00682851?sect=X01256.)
Thus there is a need for improved methods and kits for diagnosing, assessing disease risk, treating, and preventing BV and associated conditions.